搜索结果: 'gmp t cell activator'

Using a monoclonal antibody against the microtubule-associated protein tau we compared the distribution and the biochemical maturation of this protein in hippocampal pyramidal neurons in the rat in tau and in culture. In tissue sections from mature animals tau was localized heterogeneously within neurons. It was concentrated in axons; dendrites and somata showed little or no staining. In hippocampal cultures ranging from 12 h to 4 weeks in vitro tau was present in neurons but not in glial cells,as it is in situ. Within cultured neurons,however,tau was not compartmentalized but was present throughout the dendrites,axons and somata. Immunoblotting experiments showed that the biochemical maturation of tau that occurs in situ also failed to occur in culture. The young form of tau persisted,and the adult forms did not develop. In contrast the biochemical maturation and the compartmentalization of microtubule-associated protein 2 occurred normally in hippocampal cultures. These results show that the biochemical maturation and the intraneuronal compartmentalization of these two microtubule-associated proteins are independently controlled. Despite the non-restricted distribution of tau in hippocampal neurons in culture,and despite the presence of only the immature isoform which has a lessened stimulatory effect on microtubule polymerization,axons and dendrites appear to grow normally and to exhibit appropriate functional properties. View Publication

A subset of antibodies found in cattle comprises ultralong CDR-H3 regions of up to 70 amino acids. Interestingly,this type of immunoglobulin usually pairs with the single germline VL gene,V30 that is typically very conserved in sequence. In this work,we have engineered ultralong CDR-H3 common light chain bispecific antibodies targeting Epidermal Growth Factor Receptor (EGFR) on tumor cells as well as Natural Cytotoxicity Receptor NKp30 on Natural Killer (NK) cells. Antigen-specific common light chain antibodies were isolated by yeast surface display by means of pairing CDR-H3 diversities following immunization with a single V30 light chain. After selection,EGFR-targeting paratopes as well as NKp30-specific binders were combined into common light chain bispecific antibodies by exploiting the strand-exchange engineered domain (SEED) technology for heavy chain heterodimerization. Biochemical characterization of resulting bispecifics revealed highly specific binding to the respective antigens as well as simultaneous binding to both targets. Most importantly,engineered cattle-derived bispecific common light chain molecules elicited potent NK cell redirection and consequently tumor cell lysis of EGFR-overexpressing cells as well as robust release of proinflammatory cytokine interferon-$\gamma$. Taken together,this data is giving clear evidence that bovine bispecific ultralong CDR-H3 common light chain antibodies are versatile for biotechnological applications. View Publication

In vitro cultures of primary human airway epithelial cells (hAECs) grown at air-liquid interface have become a valuable tool to study airway biology under normal and pathologic conditions,and for drug discovery in lung diseases such as cystic fibrosis (CF). An increasing number of different differentiation media,are now available,making comparison of data between studies difficult. Here,we investigated the impact of two common differentiation media on phenotypic,transcriptomic,and physiological features of CF and non-CF epithelia. Cellular architecture and density were strongly impacted by the choice of medium. RNA-sequencing revealed a shift in airway cell lineage; one medium promoting differentiation into club and goblet cells whilst the other enriched the growth of ionocytes and multiciliated cells. Pathway analysis identified differential expression of genes involved in ion and fluid transport. Physiological assays (intracellular/extracellular pH,Ussing chamber) specifically showed that ATP12A and CFTR function were altered,impacting pH and transepithelial ion transport in CF hAECs. Importantly,the two media differentially affected functional responses to CFTR modulators. We argue that the effect of growth conditions should be appropriately determined depending on the scientific question and that our study can act as a guide for choosing the optimal growth medium for specific applications. View Publication

BACKGROUND: Cancer stem cells (CSCs) are purported to be epithelial tumour cells expressing CD44(+)CD24(lo) that exhibit aldehyde dehydrogenase activity (Aldefluor(+)). We hypothesised that if CSCs are responsible for tumour dissemination,disseminated cells in the bone marrow (BM) would be positive for putative breast CSC markers. Therefore,we assessed the presence of Aldefluor(+) epithelial (CD326(+)CD45(dim)) cells for the presence of the CD44(+)CD24(lo) phenotype in BM of patients with primary breast cancer (PBC). METHODS: BM aspirates were collected at the time of surgery from 66 patients with PBC. Thirty patients received neoadjuvant chemotherapy (NACT) prior to aspiration. BM was analysed for Aldefluor(+) epithelial cells with or without CD44(+)CD24(lo) expression by flow cytometry. BM aspirates from three healthy donors (HD) were subjected to identical processing and analyses and served as controls. RESULTS: Patients with triple-receptor-negative (TN) tumours had a significantly higher median percentage of CD44(+)CD24(lo) CSC within Aldefluor(+) epithelial cell population than patients with other immunohistochemical subtypes (P=0.018). Patients with TN tumours or with pN2 or higher pathologic nodal status were more likely to have a proportion of CD44(+)CD24(lo) CSC within Aldefluor(+) epithelial cell population above the highest level of HD. Furthermore,patients who received NACT were more likely to have percentages of Aldefluor(+) epithelial cells than the highest level of HD (P=0.004). CONCLUSION: The percentage of CD44(+)CD24(lo) CSC in the BM is higher in PBC patients with high risk tumour features. The selection or enrichment of Aldefluor(+) epithelial cells by NACT may represent an opportunity to target these cells with novel therapies. View Publication

Mucopolysaccharidosis type II (MPS II) is an X-linked lysosomal storage disorder resulting from deficiency of iduronate-2-sulphatase activity. The disease manifests almost exclusively in males; only 16 symptomatic heterozygote girls have been reported so far. We describe the results of X-chromosome inactivation analysis in a 5-year-old girl with clinically severe disease and heterozygous mutation p.Arg468Gln in the IDS gene. X inactivation analysed at three X-chromosome loci showed extreme skewing (96/4 to 99/1) in two patient's cell types. This finding correlated with exclusive expression of the mutated allele. Induced pluripotent stem cells (iPSC) generated from the patient's peripheral blood demonstrated characteristic pluripotency markers,deficiency of enzyme activity,and mutation in the IDS gene. These cells were capable of differentiation into other cell types (cardiomyocytes,neurons). In MPS II iPSC clones,the X inactivation ratio remained highly skewed in culture conditions that led to partial X inactivation reset in Fabry disease iPSC clones. Our data,in accordance with the literature,suggest that extremely skewed X inactivation favouring the mutated allele is a crucial condition for manifestation of MPS II in females. This suggests that the X inactivation status and enzyme activity have a prognostic value and should be used to evaluate MPS II in females. For the first time,we show generation of iPSC from a symptomatic MPS II female patient that can serve as a cellular model for further research of the pathogenesis and treatment of this disease. View Publication

There is potential interest for combining allogeneic hematopoietic cell transplantation (HCT),and particularly allogeneic HCT with a nonmyeloablative regimen,to the tyrosine kinase inhibitor imatinib (Glivec; Novartis,Basel,Switzerland,http://www.novartis.com) in order to maximize anti-leukemic activity against Philadelphia chromosome-positive leukemias. However,because imatinib inhibits c-kit,the stem cell factor receptor,it could interfere with bone marrow engraftment. In this study,we examined the impact of imatinib on normal progenitor cell function. Imatinib decreased the colony-forming capacity of mobilized peripheral blood human CD133(+) cells but not that of long-term culture-initiating cells. Imatinib also decreased the proliferation of cytokine-stimulated CD133(+) cells but did not induce apoptosis of these cells. Expression of very late antigen (VLA)-4,VLA-5,and CXCR4 of CD133(+) cells was not modified by imatinib,but imatinib decreased the ability of CD133(+) cells to migrate. Finally,imatinib did not decrease engraftment of CD133(+) cells into irradiated nonobese diabetic/severe combined immunodeficient/beta2m(null) mice conditioned with 3 or 1 Gy total body irradiation. In summary,our results suggest that,despite inhibition of hematopoietic progenitor cell growth in vitro,imatinib does not interfere with hematopoietic stem cell engraftment. View Publication

The derivation of three-dimensional (3D) stratified neural retina from pluripotent stem cells has permitted investigations of human photoreceptors. We have generated a H9 human embryonic stem cell subclone that carries a green fluorescent protein (GFP) reporter under the control of the promoter of cone-rod homeobox (CRX),an established marker of postmitotic photoreceptor precursors. The CRXp-GFP reporter replicates endogenous CRX expression in vitro when the H9 subclone is induced to form self-organizing 3D retina-like tissue. At day 37,CRX+ photoreceptors appear in the basal or middle part of neural retina and migrate to apical side by day 67. Temporal and spatial patterns of retinal cell type markers recapitulate the predicted sequence of development. Cone gene expression is concomitant with CRX,whereas rod differentiation factor neural retina leucine zipper protein (NRL) is first observed at day 67. At day 90,robust expression of NRL and its target nuclear receptor NR2E3 is evident in many CRX+ cells,while minimal S-opsin and no rhodopsin or L/M-opsin is present. The transcriptome profile,by RNA-seq,of developing human photoreceptors is remarkably concordant with mRNA and immunohistochemistry data available for human fetal retina although many targets of CRX,including phototransduction genes,exhibit a significant delay in expression. We report on temporal changes in gene signatures,including expression of cell surface markers and transcription factors; these expression changes should assist in isolation of photoreceptors at distinct stages of differentiation and in delineating coexpression networks. Our studies establish the first global expression database of developing human photoreceptors,providing a reference map for functional studies in retinal cultures. View Publication

Retinoic acid (RA) is used to treat leukemia and other cancers through its ability to promote cancer cell differentiation. Strategies to enhance the anticancer effects of RA could deepen and broaden its beneficial therapeutic applications. In this study,we describe a receptor cross-talk system that addresses this issue. RA effects are mediated by RAR/RXR receptors that we show are modified by interactions with the aryl hydrocarbon receptor (AhR),a protein functioning both as a transcription factor and a ligand-dependent adaptor in an ubiquitin ligase complex. RAR/RXR and AhR pathways cross-talk at the levels of ligand-receptor and also receptor-promoter interactions. Here,we assessed the role of AhR during RA-induced differentiation and a hypothesized convergence at Oct4,a transcription factor believed to maintain stem cell characteristics. RA upregulated AhR and downregulated Oct4 during differentiation of HL-60 promyelocytic leukemia cells. AhR overexpression in stable transfectants downregulated Oct4 and also decreased ALDH1 activity,another stem cell-associated factor,enhancing RA-induced differentiation as indicated by cell differentiation markers associated with early (CD38 and CD11b) and late (neutrophilic respiratory burst) responses. AhR overexpression also increased levels of activated Raf1,which is known to help propel RA-induced differentiation. RNA interference-mediated knockdown of Oct4 enhanced RA-induced differentiation and G(0) cell-cycle arrest relative to parental cells. Consistent with the hypothesized importance of Oct4 downregulation for differentiation,parental cells rendered resistant to RA by biweekly high RA exposure displayed elevated Oct4 levels that failed to be downregulated. Together,our results suggested that therapeutic effects of RA-induced leukemia differentiation depend on AhR and its ability to downregulate the stem cell factor Oct4. View Publication

BACKGROUND The Activin A and bone morphogenetic protein (BMP) pathways are critical regulators of the immune system and of bone formation. Inappropriate activation of these pathways,as in conditions of congenital heterotopic ossification,are thought to activate an osteogenic program in endothelial cells. However,if and how this occurs in human endothelial cells remains unclear. METHODS We used a new directed differentiation protocol to create human induced pluripotent stem cell (hiPSC)-derived endothelial cells (iECs) from patients with fibrodysplasia ossificans progressiva (FOP),a congenital disease of heterotopic ossification caused by an activating R206H mutation in the Activin A type I receptor (ACVR1). This strategy allowed the direct assay of the cell-autonomous effects of ACVR1 R206H in the endogenous locus without the use of transgenic expression. These cells were challenged with BMP or Activin A ligand,and tested for their ability to activate osteogenesis,extracellular matrix production,and differential downstream signaling in the BMP/Activin A pathways. RESULTS We found that FOP iECs could form in conditions with low or absent BMP4. These conditions are not normally permissive in control cells. FOP iECs cultured in mineralization media showed increased alkaline phosphatase staining,suggesting formation of immature osteoblasts,but failed to show mature osteoblastic features. However,FOP iECs expressed more fibroblastic genes and Collagen 1/2 compared to control iECs,suggesting a mechanism for the tissue fibrosis seen in early heterotopic lesions. Finally,FOP iECs showed increased SMAD1/5/8 signaling upon BMP4 stimulation. Contrary to FOP hiPSCs,FOP iECs did not show a significant increase in SMAD1/5/8 phosphorylation upon Activin A stimulation,suggesting that the ACVR1 R206H mutation has a cell type-specific effect. In addition,we found that the expression of ACVR1 and type II receptors were different in hiPSCs and iECs,which could explain the cell type-specific SMAD signaling. CONCLUSIONS Our results suggest that the ACVR1 R206H mutation may not directly increase the formation of mature chondrogenic or osteogenic cells by FOP iECs. Our results also show that BMP can induce endothelial cell dysfunction,increase expression of fibrogenic matrix proteins,and cause differential downstream signaling of the ACVR1 R206H mutation. This iPSC model provides new insight into how human endothelial cells may contribute to the pathogenesis of heterotopic ossification. View Publication

Central issues in intracoronary infusion (ICI) of bone marrow (BM)-cells to damaged myocardium for improving cardiac function are the cell number that is feasible and safe to be administrated as well as the retention of cells in the target area. Our study addressed these issues in eight patients with chronic ischemic cardiomyopathy undergoing ICI of selected BM-progenitors. We could immunomagnetically isolate 0.8 +/- 0.32 x 10(7) CD133(+) cells and 0.75 +/- 0.24 x 10(7) CD133(-)CD34(+) cells from 310 +/- 40 ml BM. After labeling these cells with (99m)Tc-hexamethylpropylenamineoxime,they were infused into the infarct-related artery without any complication. Scintigraphic images 1 (eight patients) and 24 hours (four patients) after ICI revealed an uptake of 9.2% +/- 3.6 and 6.8% +/- 2.4 of the total infused radioactivity in the infarcted area of the heart,respectively; the remaining activity was distributed mainly to liver and spleen. We conclude that through ICI of CD133(+) and CD133(-)CD34(+) BM-progenitors a significant number of them are preferentially attracted to and retained in the chronic ischemic myocardium. View Publication

In humans,recent clinical and experimental data from hematopoietic stem cell transplantation revealed that donor-derived alloreactive NK cells exert a beneficial graft versus leukemia effect. The existence of donor-derived alloreactive NK cells can be predicted on the basis of donor killer cell Ig-like receptor (KIR) gene profile and HLA class I typing of both donor and recipient. Moreover,the size of the alloreactive NK cell population can be directly assessed by the combined use of anti-KIR-specific mAb. In this study,in an attempt to improve the definition of alloreactive NK cell subsets,we assessed the KIR genotype and phenotype in a cohort of 44 donors. This approach allowed the identification of two different KIR2DL3 alleles (KIR2DL3*005 and the novel allele KIR2DL3*015) that did not react with the anti-KIR2DL3-specific ECM41 mAb. In contrast,both alleles were recognized at the cell surface by several mAb reacting with KIR2DL2/L3/S2. Notably,KIR2DL3*005 was also stained by the anti-KIR2DL1/S1-specific EB6B and 11PB6 mAb. Functional analysis revealed that,despite its particular mAb reactivity,the specificity of KIR2DL3*005 for HLA-C molecules did not differ from that of other KIR2DL2/L3 alleles. Finally,site-directed mutagenesis demonstrated that glutamine at position 35 is required for ECM41 staining,whereas glutamic acid 35 and arginine 50 are relevant for staining with EB6B or 11PB6 mAb. Our present data represent a substantial progress in the characterization of the NK cell repertoire and an improved phenotypic/functional definition of given KIR(+) subsets. View Publication

To further clarify the transformation from monoclonal gammopathy of undetermined significance (MGUS) to plasma cell myeloma (PCM),we compared interphase fluorescence in situ hybridization (FISH) patterns in 381 MGUS and 301 PCM patients. According to the World Health Organization and the International Myeloma Working Group,a threshold of 10% of bone marrow plasma cells separated MGUS from PCM. After magnetic activated cell sorting for CD138(+) cells,FISH succeeded in 272 of 301 (90.4%) PCM,but in only 302 of 381 (79.3%) MGUS cases (P textless 0.001). Cytogenetic alterations were more frequent in PCM (237 of 272; 87.1%) than MGUS (169 of 302; 56.0%; P = 0.0002). PCM showed a median of two cytogenetic alterations (range,0-9) and MGUS one (range,0-6). Considering only cases with a yield of plasma cells allowing five or more FISH probes,del(13)(q14) was found in 99 of 251 (39.3%) PCM but in only 59 of 267 (22.1%) MGUS (P = 0.0001),del(17p) in 15 PCM (6.0%) and in 6 MGUS (2.2%) patients (P = 0.029). A t(4;14)/IGH-FGFR3 was detected in 28 PCM (11.1%) and 5 MGUS (1.9%; P textless 0.001). The t(11;14)/IGH-CCND1 and the t(14;16)/IGH-MAF showed no significant differences. Cytomorphology detected higher numbers of plasma cells than multiparameter flow cytometry (median ratio 4.25). This study underlines the genetic heterogeneity of MGUS similar to PCM. Genetic analysis might contribute to more diversified monitoring strategies for MGUS patients. View Publication